Overcoming radioresistance is essential for improving radiotherapy. Recently, we found that miR-21 as an onco-miR involved cell radioresistance through promoting DNA double strand repair. These results suggest that targeting miR-21 is a good strategy for sensitizing tumor cells to radiation; however, since radiation could induce miR-21 up-regulation, the up-regulated miR- 21 could in turn reduce the effects of targeting miR-21 alone on radiosensitization. In addition, the miR-21 status in the surrounding tumor tissue may affect tumor response to ionizing radiation treatment since the tumor microenvironment is an important factor that affects the tumor situation. Therefore, in order to efficiently block the function of miR-21 in tumor radioresistance, we need to know the whole picture of miR-21 signaling in irradiated tumor cells in vitro and in vivo. For this purpose, we designed two aims in this proposal. Aim 1: Determine how IR stimulates miR-21 up-regulation. We will elucidate IR-promoted upstream regulation of miR-21 with the methods and approaches of molecular and cellular biology by answering the following four questions: 1) Whether the IR-activated ATR stimulates miR-21 expression through phosphorylating STAT3 at S694. 2) Whether STAT3 as a transcription factor could directly stimulate the EGFR expression to respond to IR. 3) Whether EGFR inhibits miR-21 maturation through phosphorylating AGO2 at Y393 to respond to IR. 4) Whether miR-21 could directly target p53 that negatively regulates EGFR expression through inhibiting YY1 and SP1 (the transcription factors of EGFR). Aim 2: Determine whether and how miR-21 in the tumor microenvironment affects the tumor response to IR treatment. We will use two mouse models: 1) miR-21 knock-in or knockout mouse model: we will use tumor cells that can grow tumors in these immune efficient mice. 2) nude mouse model: we will use the human tumor cells that can grow xenograft tumors in the immune deficient mice. These tumor cell lines (mouse or human) will be down-regulated with miR-21 or its upstream regulators, subcutaneously inoculated into the hind legs of the mice. We will observe the tumor size and examine the level/activity of miR- 21/its relevant regulators in the tumor tissue and the surrounding tissues after the tumor areas are irradiated. The results from this proposal are expected to help us better understand the role of miR-21 in mediating tumor radioresistance in vitro and in vivo, thus, providing an efficient way to block the miR-21 pathway for improving radiotherapy in the near future.